The anaerobe Porphyromonas gingivalis (P. gingivalis) has been implicated as a major causative organism of adult onset periodontal disease. Enzymes from this organism have been found to degrade several proteins, including, for example, collagen, fibrinogen, immunoglobulins, complement proteins, and fibronectin Recent evidence has shown that three proteinases released from P. gingivalis may have a physiological role in modulating the human immune system in addition to their general ability to degrade proteins. These three proteinases are referred to as gingipains and include arginine-specific gingipain A (RgpA) and arginine-specific gingipain B (Rgp B), which are capable of specifically cleaving after arginine residues, and lysine-specific gingipain (Kgp) which is able to specifically cleave after lysine residues. Working in concert, these proteinases have been shown to produce bradykinin from high molecular weight kininogen, either directly or indirectly (kallikrein activation), resulting in the enhancement of vascular permeability (Imamura, et al., Infect, Immun., 63(5):1999-2003 (1995)). This mechanism, which is used to provide nutritional components for the growth and proliferation of P. gingivalis, is presumed to be responsible for both the increased gingival crevicular fluid (GCF) and edema clinically noted in periodontal pockets of patients with advanced periodontitis (Darany et al., J. Periodontol., 63:743-747 (1992)).
The interaction of P. gingivalis with a host""s immune system response has been paradoxical, in that P. gingivalis has demonstrated both pro-inflammatory and anti-inflammatory responses. For example, P. gingivalis lipopolysaccharide has been shown to increase mRNA levels of interleukin-8 (IL-8) in neutrophils (Sugita et al., Inflammation, 2(3):253-267(1998)), and gingipains R have been shown to increase neutrophil chemotaxis by release of C5a from C5 of the complement system (Wingrove et al., J. Biol. Chem., 7(26):18902-18907 (1992)). However, these proteinases are also capable of cleaving the C5a receptor from infiltrating neutrophils (Jagels et al., Infect. Immun., 64(6):198-1991 (1996)), thereby effectively neutralizing localized chemotactic activity. Additionally, P. gingivalis has the ability to inhibit both IL-8 accumulation in gingival epithelial cells (Darveau et al., Infect. Immun., 66(4):1660-1665 (1998)), as well as transepithelial migration (Madianos et al., Infect, Immun., 65(10):3983-3990 (1997)).
These apparent activity contradictions may potentially be explained by the compartmentalization of the oral cavity, wherein distal activation of chemotactic components and proximal paralysis of these factors creates a xe2x80x9cleukocyte wallxe2x80x9d between the periodontal plaque and gingival epithelium (Miyasaki, J. Periodontol., 2:761-774 (1991)). Indeed, it has been reported that _soluble gingipains can stimulate IL-8 activity, whereas membrane bound gingipains, with a limited ability to diffuse beyond the plaque surface, completely degrade IL-8 (Mikolajczyk-Pawlinska et al., FEBS Lett., 440:282-286 (1998)).
The recruitment of neutrophils to the xe2x80x9cleukocyte wallxe2x80x9d through both the increased leakage of blood vessels and a chemotactic gradient at first appear to be suicidal to P. gingivalis. However, such a scenario is not likely, as P. gingivalis is known to have evolved mechanisms to survive in the presence of neutrophils. For example, P. gingivalis proteinases have been shown to degrade C3 complement and immunoglobulins (Schenkein et al., J. Immunol., 154:5331-5337 (1995)), thereby averting opsonization and subsequent detection by a host. Furthermore, gingipain R has been shown to have an inhibitory effect on the oxidative burst utilized by neutrophils to kill microorganisms (Kadowaki et al., J. Biol. Chem., 269(33):21371-21378 (1994)). Similarly, the bacterial outer membrane of P. gingivalis may function as an antioxidant sink due to the incorporation of large amounts of heme (Smalley et al., J. Biochem., 331:681-685 (1998)).
Activated neutrophils in the leukocyte wall typically undergo degranulation due to the inability to phagocytize foreign organisms, thereby expelling large quantities of the proteinases human neutrophil elasase (HNE) and cathepsin G. Although these proteinases may cause abnormal connective tissue destruction, the presence of human plasma proteinase inhibitors (serpins) typically minimize connective tissue destruction by complexing with endogenous proteinases. These complexes are ultimately absorbed by the liver for degradation. For example, high protein levels of the xcex11-proteinase inhibitor (xcex11-PI) have been detected in GCF samples from patients diagnosed with severe periodontal disease (Huynh et al., J. Clin. Periodontol., 19:187-192 (1992)). However, despite the presence of xcex11-PI a high HNE activity is observed indicating that the xcex11-PI must be present in either complexed, oxidized, or proteolytically inactivated forms (Uitto et al., J. Clin. Periodontol., 2:30-37 (1996)). This observation is supported by evidence showing that less than 35% of available xcex11-PI in the GCF is active as an inhibitor (Smith et al., Archs. Oral Biol., 22(4):301-306 (1994)). Additionally, it has been shown that patients with xcex11-PI deficiencies have a significantly higher frequency of periodontal pocket depths xe2x89xa75 mm, thereby being predisposed to manifestations of periodontal disease (Fokkems et al., J. Clin. Periodontol., 25:617-623 (1998)). Thus, there is a need for further understanding the interaction of such serpins with other oral bacterial proteinases.
Described herein are the isolation, purification and characterization of a polypeptide, particularly an oral bacterial polypeptide that interacts with a serpin such as the human serpin xcex11-PI. This polypeptide is referred to as xe2x80x9cperiodontain,xe2x80x9d not only because of its function as a proteinase, but also because this polypeptide may function as a putative factor in the dysregulation of serpin function in the periodontal cavity of an animal. Additionally, the deduced amino acid sequence of periodontain, as determined by both partial peptide sequencing of the purified polypeptide and characterization of the P. gingivalis genome is provided.
Accordingly, the present invention provides an isolated oral bacterial polypeptide which has amidolytic activity for cleavage of a nondenatured human (xcex11-proteinase inhibitor at a reactive site loop region of the inhibitor. The isolated polypeptide demonstrates amidolytic activity in a solution containing about 1 nM to about 500 mM Tris, about 500 xcexcM to about 100 mM cysteine maintained at a pH of about 7 to about 8. Preferably, the polypeptide is isolated from Porphyromonas gingivalis and is a cysteine proteinase.
The polypeptide of the invention preferably has a molecular weight of about 70 kD to about 80 kD as determined by gel filtration. The polypeptide of the invention will preferably cleave the reactive site loop region of the inhibitor represented by SEQ ID NO: 4 between glutamine and alanine and also between phenylalanine and leucine.
The present invention also provides an isolated polypeptide that is an oral bacterial cysteine proteinase and has amidolytic activity for cleavage of a nondenatured serpin at a reactive site loop region of the serpin. The isolated polypeptide is preferably isolated from Porphyromonas gingivalis. The isolated polypeptide of the invention further has the capability to cleave a target polypeptide nonspecifically.
The present invention further provides an isolated polypeptide that is isolated from Porphyromonas gingivalis and has amidolytic activity for cleavage of a nondenatured serpin at a reactive site loop region of the serpin.
The isolated polypeptide preferably contains an amino acid sequence having a percentage amino acid identity of greater than 37% to that of amino acid 148 to amino acid 843 of SEQ ID NO: 1. More preferably, the isolated polypeptide contains an amino acid sequence having a percentage amino acid identity of greater than 52% to that of amino acid 148 to amino acid 629 of SEQ ID NO: 1. In particularly preferred embodiments, the isolated polypeptide has an amino acid sequence represented by SEQ ID NO: 1, an active analog or an active fragment thereof, and more preferably, an amino acid sequence represented by amino acid 148 to amino acid 843 of SEQ ID NO: 1, an active analog or an active fragment thereof.
The present invention farther provides an isolated nucleic acid encoding an oral bacterial polypeptide which has amidolytic activity for cleavage of a nondenatured human xcex11-proteinase inhibitor at a reactive site loop region of the inhibitor. Also provided is an isolated nucleic acid encoding a polypeptide which is an oral bacterial cysteine proteinase and has amidolytic activity for cleavage of a nondenatured serpin at a reactive site loop region of the serpin. The present invention further provides an isolated nucleic acid encoding a polypeptide which is isolated from Porphyromonas gingivalis and has amidolytic activity for cleavage of a nondenatured serpin at a reactive site loop region of the serpin.
The isolated nucleic acid fragment of the invention preferably has a nucleotide sequence represented by SEQ ID NO: 2. The isolated nucleic acid fragment of the invention further preferably encodes a polypeptide having an amino acid sequence with a percentage amino acid identity of greater than 37% when compared to amino acid 148 to amino acid 843 of SEQ ID NO: 1.
The present invention also provides an isolated nucleic acid fragment as described herein wherein the complement of the nucleic acid fragment hybridizes to SEQ ID NO: 2 under hybridization conditions of 0.5 M phosphate buffer, pH 7.2, 7% sodium dodecyl sulfate (SDS), 10 mM ethylenediaminetetra-acetate (EDTA), at 68xc2x0 C., followed by three 20 minute washes in 2xc3x97SSC (1xc3x97SSC is 0.15 M NaCl, 0.015 M sodium citrate), 0.1% SDS, at 65xc2x0 C., wherein at least about 15 nucleotides of the complement hybridize.
The present invention further provides a method for identifying an inhibitor of a polypeptide which has amidolytic activity for cleavage of a nondenatured serpin at a reactive site loop region of a serpin. The method includes isolating an agent that inhibits the amidolytic activity of the polypeptide by incubating the polypeptide with the agent under conditions that promote amidolytic activity of the polypeptide and determining if the amidolytic activity of the polypeptide is reduced relative to the amidolytic activity of the polypeptide in the absence of the agent. Preferably, the polypeptide is isolated form Porphyromonas gingivalis. 
The invention also provides an immunogenic composition having a polypeptide which has amidolytic activity for cleavage of a nondenatured serpin at a reactive site loop region of a serpin and is capable of eliciting antibodies in an animal.
Also provided is a composition containing an inhibitor to a polypeptide that has amidolytic activity for cleavage of a nondenatured serpin at a reactive site loop region of a serpin isolated from an oral bacterium.
Definitions
xe2x80x9cPolypeptidexe2x80x9d as used herein refers to a polymer of amino acids and does not refer to a specific length of a polymer of amino acids. Thus, for example, the terms peptide, oligopeptide, protein, and enzyme are included within the definition of polypeptide. This term also includes post-expression modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations, and the like.
xe2x80x9cProteinase,xe2x80x9d xe2x80x9cpeptidase,xe2x80x9d and xe2x80x9cproteasexe2x80x9d all refer to enzymes that catalyze the hydrolysis of peptide bonds in a polypeptide. A xe2x80x9cpeptide bondxe2x80x9d or xe2x80x9camide bondxe2x80x9d is a covalent bond between the alpha-amino group of one amino acid and the alpha-carboxyl group of another amino acid.
As used herein, the term xe2x80x9cisolatedxe2x80x9d means that a polypeptide is either removed from its natural environment or synthetically derived. Preferably, the polypeptide is purified, i.e., essentially free from any other polypeptides and associated cellular products or other impurities.
xe2x80x9cAmidolytic activityxe2x80x9d refers to the ability of a polypeptide to catalyze the hydrolysis of at least one peptide bond in another polypeptide. The term xe2x80x9ccleavagexe2x80x9d can also be used to refer to the hydrolysis of a peptide bond in a polypeptide.
xe2x80x9cPeriodontainxe2x80x9d refers to a polypeptide having amidolytic activity with respect to a target polypeptide. Preferably, the polypeptide is a cysteine proteinase that is isolated from an oral bacteria, such as, P. gingivalis. A xe2x80x9ctarget polypeptide,xe2x80x9d as used herein, includes any denatured polypeptide and/or a native polypeptide, i.e., nondenatured, that contains at least one exposed contiguous amino acid region, such as a random coil as determined for example, by x-ray crystallography, that is susceptible to cleavage, preferably by periodontain. The exposed amino acid region typically contains at least about 3 contiguous amino acids, and preferably at least about 10 contiguous amino acids. Periodontain target polypeptides include, for example, serpin polypeptides in general and mammalian serpin polypeptides specifically. A preferred target polypeptide of the invention is the human serpin, xcex11-proteinase inhibitor (xcex11-PI) (SEQ ID NO: 3). With respect to a serpin polypeptide, the exposed amino acid region, e.g., random coil, as used herein, is referred to as a xe2x80x9creactive site loopxe2x80x9d region or xe2x80x9cRSL.xe2x80x9d The RSL region of human xcex11-PI is about 15 contiguous amino acids (SEQ ID NO: 4).
An xe2x80x9cactive analogxe2x80x9d or xe2x80x9cactive fragmentxe2x80x9d of a polypeptide of the invention is one that has amidolytic activity by hydrolysis of a peptide bond present in the target polypeptide as described herein. Active analogs and active fragments are described in greater detail herein.
xe2x80x9cNucleic acid fragmentxe2x80x9d as used herein refers to a linear polymeric form of nucleotides of any length, either ribonucleotides or deoxynucleotides, and includes both double- and single-stranded DNA and RNA. A nucleic acid fragment may include both coding and noncoding regions that can be obtained directly from a natural source (e.g., a microorganism), or can be prepared with the aid of recombinant or synthetic techniques. A nucleic acid molecule may be equivalent to this nucleic acid fragment or it can include this fragment in addition to one or more other nucleotides or polynucleotides. For example, the nucleic acid molecule of the invention can be a vector, such as an expression or cloning vector.
xe2x80x9cPercentage amino acid identityxe2x80x9d refers to a comparison of the amino acids of two polypeptides as described herein. Amino acid alignment may be determined, for example, using the sequence alignment program CLUSTAL W available at www.genome.ad.ip/SIT/CLUSTALW.html. and percent amino acid identity may be determined by BLAST 2 SEQUENCES at National Center for Biotechnology Information (NCBI) website: www.ncbi.nlm.nih.gov.